Our laboratory is working in the area of drug resistance in human tumor cells. We have recently described a methotrexate resistant human breast cancer cell line (MTXR MCF-7) which contains amplified dihydrofolate reductase genes as the mechanism of resistance. We have now cloned the human genomic dihydrofolate reductase gene from this cell line as well as two non functional human DHFR pseudogene. We have also constructed a functional dihydrofolate reductase minigene using both genomic and cDNA sequences. This dihydrofolate reductase minigene has been transfected into mutant chinese hamster cells which are deficient in DHFR activity. The human DHFR minigene is able to rescue these mutant cells within a relatively high frequency (.12%). In addition, we are developing various deletion mutants of this gene in order to identify those DNA sequences which are necessary for functional expression as well the sequences which are necessary for the intracellular modulation of DHFR gene expression. Because the efficiency of transfection of this minigene is quite high in the absence of many additional viral DNA sequences added to it, we are now studying whether the human dihydrofolate reductase gene contains any DNA sequences which function as enhancer sequences. We have also studied the regulation DHFR gene expression in human breast cancer lines. These studies have shown that estrogen increases while tamoxifin decreases the expression of the DHFR gene at the level of transcription. Other studies have confirmed that methotrexate (MTX) also induces DHFR levels and the mechanism this regulation is currently under investigation. We have also developed another methotrexate resistant human breast cancer cell line in our lab with multiple defects associated with drug resistant.